QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jiménez-Cervantes, C. Articles by García-Borrón, J. C. Search for Related Content PubMed PubMed Citation Articles by Jiménez-Cervantes, C. Articles by García-Borrón, J. C. Social Bookmarking                         What's this?

Inhibition of melanogenesis in response to oxidative stress: transient downregulation of melanocyte differentiation markers and possible involvement of microphthalmia transcription factor

Department of Biochemistry and Molecular Biology, School of Medicine, University of Murcia, Apto 4021, Campus de Espinardo, 30100 Murcia, Spain

View larger version (15K): [in a new window]   Fig. 1. Inhibition of the tyrosine hydroxylase activity of mouse and human melanoma cells by H2O2. B16 mouse melanoma and SCL and BEU human melanoma cells were exposed to 1 mM H2O2 for 20 minutes, and then allowed to recover in H2O2-free medium for 8 or 16 hours. Cells were harvested, solubilized in 1% Igepal CA-630 and the tyrosine hydroxylase and protein content of the samples was analyzed. Results are expressed as mean±s.d. of three independent experiments. The tyrosine hydroxylase-specific activities (in µU/mg protein) of controls, treated as the 8 hours time point, were: 79±27, 170±22 and 303±27, for BEU, SCL and B16 melanoma cells, respectively.

View larger version (16K): [in a new window]   Fig. 2. Dose dependence of H2O2 inhibition of tyrosine hydroxylase activity and effect of the length of the oxidative challenge. (A) Semiconfluent B16 melanoma cells were challenged for 20 minutes with varying concentrations of H2O2, and allowed to recover in fresh medium for 16 hours before enzyme activity and protein concentration determinations. Results shown are the mean±s.d. for three independent experiments. (B) Cells were exposed to H2O2 (0.5 mM) for the times shown and allowed to recover for 8 hours before determination of their tyrosine hydroxylase- () and dopa oxidase- () specific activities. Results are the mean±s.d. of at least three independent experiments.

View larger version (14K): [in a new window]   Fig. 3. Decreased melanin contents in B16 melanoma cells treated with H2O2. (A) Cells challenged with H2O2 (0.5 mM, 20 minutes) were allowed to recover for the times shown. After harvesting with trypsin, the cells were solubilized in 1% Igepal CA-630 and centrifuged at 20,000 g, for 30 minutes. Protein concentration was determined in the supernatant, and total melanin was measured by KOH hydrolysis of the insoluble pellet. Results are the mean±s.d. of three independent experiments. (B) Growth kinetics of H2O2-treated cells. 4x104 cells were seeded in six-well plates, in sets of triplicate wells, and incubated for 24 hours in the complete medium described in Materials and Methods. Then the cells were challenged with H2O2 (0.5 mM, 20 minutes) or with Hank’s solution, and further incubated in complete medium from 1 to 24 hours. After trypsinization, the number of viable cells was determined by Trypan Blue exclusion. Open symbols, H2O2-treated cells; closed symbols, control cells. Results are shown as mean ± s.d. of triplicate wells.

View larger version (47K): [in a new window]   Fig. 4. Decreased Tyr and Tyrp1 contents in H2O2-challenged B16 melanoma cells. (A) Cells were challenged with H2O2 (0.5 mM, 20 minutes), allowed to recover for the times shown, trypsin-harvested and solubilized. Equal amounts of protein (30 µg/lane) were electrophoresed on 9% SDS-PAGE gels and transferred to PVDF membranes. The blots were probed with PEP1 (anti-Tyrp1, first panel) and PEP7 (anti-Tyr, second panel), and stained with a chemiluminescent substrate (Amersham Pharmacia Biotech, Buckinghamshire, UK). Comparable loading and transfer was ascertained by cutting the lower portion of the blot and staining for total protein with Amido Black (lower panel). Similar trends were obtained in three independent experiments. C, control. Separate controls are included for cells grown for 16 and 24 hours after the oxidative challenge, as a density-dependent increase in melanocyte differentiation markers is often observed (Hornyak et al., 2000). (B) Densitometric quantification of immunoblots. Blots were quantified in a laser densitometer. The results shown represent the relative abundance of Tyr (white bars) and Tyrp1 (black bars), with respect to controls collected at 1 (for time points up to 8 hours), 16 and 24 hours, and are the mean±s.d. for three experiments.

View larger version (16K): [in a new window]   Fig. 5. Levels of total glutathione and the lipid peroxidation marker MDA in H2O2-treated cells. Cells were pulsed with H2O2 (0.5 mM, 20 minutes), and allowed to recover in H2O2-free complete medium for up to 24 hours. (A) Evolution of total glutathione during recovery after oxidative challenge. H2O2-pulsed cells were harvested by trypsin treatment. An aliquot was saved for solubilization and protein determination. The remaining cell suspension was centrifuged and solubilized in 131 mM 5-sulfosalicylic acid for determination of total, reduced and oxidized glutathione. Results are shown as % values with respect to control cells, harvested with the 5 minutes time point, whose glutathione contents was 29±13 pmol/µg protein, and are the mean±s.d. for at least three independent experiments. (B) Lipid peroxidation in H2O2-challenged cells. Cells were treated with 0.5 mM H2O2 (20 minutes), and allowed to recover for the times shown before determination of their MDA contents. Results are expressed as % of controls treated in parallel, but without H2O2 and are the mean±s.d. for three independent experiments.

View larger version (17K): [in a new window]   Fig. 6. Kinetics of downregulation of the melanogenic activities and recovery of basal activity levels 48 hours after the oxidative challenge. Cells were pulsed for 20 minutes with H2O2 (0.5 mM) and allowed to recover in complete medium for up to 48 hours. The dopa oxidase () and tyrosine hydroxylase () activities of solubilized extracts were measured. The results shown are the mean±s.d. for three independent experiments. Controls for calculation of the % residual activity were collected with the 8, 16, 24 and 48 hours time points. The inset shows the changes in tyrosinase protein levels at 8 and 48 hours, assessed by western blot with PEP7 as primary antibody, after separation by 12% SDS-PAGE.

View larger version (19K): [in a new window]   Fig. 7. Northern blot analysis of mRNA levels for five melanocyte differentiation markers in H2O2-challenged B16 melanoma cells. (A) Representative blots, performed with 10 µg of total RNA from control cells (C), treated exactly as the 8 hours time point or cells treated with H2O2 (0.5 mM, 20 minutes) and allowed to recover for 8 or 16 hours. The blots were probed for Tyr, Dct, Tyrp1, silver, Mc1r and ODC mRNA, as indicated, as well as for GAPDH (shown below each lane). Blots were performed in triplicate for Tyr, Dct, Tyrp1 and ODC, and in duplicate for the silver locus product and for Mc1r. (B) Quantification of mRNA variations. Northern blots were quantified by phosphorimaging, in a BioRad GS-525 Molecular Imager. The results, corrected for loading by comparison to the GAPDH signal, are shown as % expression with respect to the control untreated cells, and are the mean±s.d. for the mRNA species analyzed in triplicate (Tyr, Dct, Tyrp1 and ODC). For these species, the statistical significance of the variations was analyzed by calculating two-tailed P values by means of an unpaired Student’s t-test, using the Prism software. , P<0.01. For the species analyzed in duplicate (silver and Mc1r), the values given are the mean±range for the two determinations, and no statistical analysis was performed.

View larger version (32K): [in a new window]   Fig. 8. Downregulation of Mitf mRNA levels in B16 melanoma cells following oxidative stress. (A) B16 cells were challenged with 0.5 mM H2O2 for 20 minutes and allowed to recover in complete medium for up to 8 hours. For TPA treatment, the agent was kept in the medium at a final concentration of 40 nM throughout the experiment, and cells were incubated for times ranging from 0.5 to 8 hours, as indicated on top of each lane. The levels of Mitf mRNA were analyzed by Northern blot. GAPDH mRNA was also analyzed for normalization. C, control untreated cells. Note that the maximal downregulation of Mitf mRNA is similar for TPA and H2O2 treatments. Similar trends were obtained in two independent experiments. (B) Quantification of Mitf mRNA variations. Blots were quantified as described in Fig. 7. The results shown correspond to % expression with respect to control cells, and are the mean±range for two determinations. (C) B16 cells were stimulated with the superpotent MSH analogue [Nle4, D-Phe7]-MSH (Sigma, St Louis, MO), at a final concentration of 100 nM, or with the adenylate cyclase stimulator forskolin (10 µM). Cells were harvested 2 hours after addition of the agents, and Mitf expression was analyzed by Northern blot. C, control; M, MSH-treated cells; F, forskolin-treated cells. (D) Semiquantitative RT-PCR analysis of Mitf mRNA levels in H2O2-challenged B16 cells. Cells were treated with H2O2, as in A, and allowed to recover for 1, 2, 4 and 8 hours, as indicated. Total RNA was extracted, and cDNA was prepared. Equivalent amounts of cDNA were amplified with primers specific for Mitf and Gapdh, as a control for comparable loading of target cDNA. The reaction mixtures were analyzed by agarose gel electrophoresis. C, control; the lane on the right shows markers of the indicated size.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter